PROJECT SUMMARY Defective clearance of apoptotic cells (AC) by macrophages (efferocytosis) contribute to many diseases including atherosclerosis. Given the therapeutic potential of genes that regulate efferocytosis, we sought to identify novel regulators of this pathway in an unbiased manner. We established a genome-wide pooled CRISPR knockout screen for efferocytosis in Cas9-overexpressing murine bone marrow-derived macrophages. Individual validation of the strongest screen hits has uncovered Wdfy3 (WD repeat and FYVE domain containing 3) as a novel regulator previously never implicated in the regulation of efferocytosis or phagocytosis. The goal of this study is to focus on Wdfy3 for in-depth mechanistic studies of its molecular mechanisms and roles in atherosclerosis. Degradation of engulfed AC requires the recruitment of a subset, but not all of the autophagy machinery for LC3 lipidation and subsequent phagosome-lysosome fusion. There remains a significant knowledge gap to identify which autophagy genes participate in the degradation of phagocytosed cargos. Those that are not required for non-selective canonical autophagy (?self-eating?) are of particular interest for their potential of therapeutic targeting for activation. Wdfy3 regulates selective autophagy for clearance of aggregated proteins, but is dispensable for non-selective canonical autophagy during starvation, making it such a candidate. Our data show that Wdfy3 knockdown reduces efferocytosis in murine and human macrophages; WDFY3 is co- localized with engulfed AC; Wdfy3 expression is increased in plaque macrophages during atherosclerosis regression; in human plaque, WDFY3 expression is correlated with M2-like macrophage markers. Within this context, we hypothesize that Wdfy3 is required for macrophage efferocytosis by regulating phagosome formation and maturation and Wdfy3-mediated efferocytosis protects against atherosclerosis in hyperlipidemic mice models. Aim 1 will determine the molecular mechanisms of Wdfy3 by addressing (A) which stage of efferocytosis is regulated by Wdfy3; (B) How Wdfy3 is recruited and participates in phagosome formation and downstream events; (C) whether and how overexpression of Wdfy3 enhances efferocytosis. Aim 2 will determine (A) whether mice lacking myeloid Wdfy3 have defective efferocytosis in dexamethasone-induced thymic apoptosis and in advanced atherosclerosis; and whether myeloid overexpression of WDFY3 will enhance in vivo efferocytosis and alleviate atherosclerosis; (B) whether human iPSC-derived macrophages with WDFY3 null mutations demonstrate defective efferocytosis and enhanced inflammation. Aim 3 will examine whether other autophagy genes among the top screen hits are also key regulators of efferocytosis and their dependence on Wdfy3. This study will (1) reveal novel fundamental mechanisms of efferocytosis regulated by Wdfy3 and the potential of Wdfy3-mediated efferocytosis as a target for pro-efferocytotic therapy in atherosclerosis, and (2) provide a broadly-applicable platform for genome-wide screen of complex functional phenotypes in primary macrophages for unbiased novel discoveries.